The ability to position and probe a single cell is of great interest in fundamental cell biology, cell-based biosensor technologies, medical diagnostics, and tissue engineering. Because critical cell-to-cell differences are lost in average bulk cell measurements, the single cell analysis with its ability to reveal the response of each individual cell under stimulation is fundamental to comprehending many biological processes and mechanisms. Patterning viable single cells on an addressable array of identical cell hosts, such as an array of microelectrodes with the same physical and chemical properties, would aid the statistical analysis of single cell behavior and cell/matrix interaction. In practical applications, particularly for screening, detection, or sensing systems, microarrays of single cells allow for rapid and inexpensive analysis, require minimal sample volume, and provide high throughput data acquisition and portability.
Cell patterning, micropatterning of living cells on substrates, has experienced a rapid growth in recent years. A number of techniques have been developed to produce micro-scale cell patterns. Examples include microcontact printing, microfluidic channels, elastomeric stencils, and elastomeric membranes, which involve the delivery of proteins/peptides to guide cell adhesion or direct deposit of cells on a substrate of a single material. Cell patterning can also be achieved by tailoring surfaces to form distinct regions that have adhesive proteins or ligands to host one or groups of cells with a background inert to protein absorption and cell adhesion. Cell patterning can be accomplished via soft lithography (see, for example, Y. Xia and G. M. Whitesides, Angew. Chem. 110 (5), 568-594, 1998), photochemistry (see, for example, L. M. Tender et al., Langmuir 12:5515-5518, 1996), and photolithography techniques (see, for example, W. Knoll et al., J. Adv. Biophys. 34:231-251, 1997). In these techniques, the patterns are formed either by generation of heterogeneous chemistry on a single material or by deposition of a second material in a certain shape and geometry followed by surface modification to form heterogeneous chemistry.
One of the important applications for the cell patterning is for the development of cell-based biosensors (CBBs), in which the patterned regions are miniaturized arrays of metal electrodes and the background is an insulate substrate material. Cell-based biosensors are generally constructed by interfacing cells to a transducer that converts cellular responses into signals detectable by electronic or optical devices. CBBs are hybrid systems of biology and device that use cells' abilities to detect, transduce, and amplify very small changes of external stimuli. Cell-based biosensors offer new opportunities for many medical applications, including biothreat detection, drug evaluation, pollutant identification, and cell type determination.
Recent years have witnessed a substantial growth in application of planar microelectrode arrays in CBBs because they can be readily interfaced with electronic, optical, or chemical detecting means. Major advantages of these sensing arrays over conventional biosensors include rapid and inexpensive analyses, smaller sample size requirement, low sample contamination, high throughput and sensitivity, and portability. Among these sensors, single-cell-based sensors are of particular interest. With an array of virtually identical single cells as sensing elements integrated with real-time data acquisition technology, single-cell-based sensors can be used to experimentally study cellular pathways without interference from other cells, thereby eliminating the uncertainty incurred by states of neighboring cells. In addition, statistical analysis of cell behavior, a topic extensively pursued in cell biology, requires closely identical cell sites, and a single-cell-based system may ideally serve the purpose.
Despite the encouraging advances made with micropatterning of living cells on substrates, patterning single cells on a microarray and retaining their viability for a prolonged period of time remain as a challenge. Single cell patterning requires an area for cell adhesion at a size comparable to an individual cell, which is typically 10 to 20 μm, to minimize the probability of a second cell attachment. However, adhesion sites of such small areas tend to suppress cell spread and thus are prone to causing cell death. It was reported that cells could be geometrically switched between growth and apoptosis. Endothelial cells cultured on single islands coated with fibronectin spread and progressed through the cell cycle when the island area was larger than approximately 40 μm×40 μm, but failed to extend and underwent apoptosis when cells were restricted to areas smaller than approximately 20 μm×20 μm.
A need exists for devices and methods for patterning single cells that allow single-cell adhesion while maintaining cellular viability for a prolonged period of time. The present invention seeks to fulfill these needs and provides further related advantages.